The goal of our proposed research is to explore the effects of targeted disruption of the nuclear orphan receptors TR2 and TR4, as well as consequences of disruption of the two genes in combination, in order to determine the physiological roles of these receptors. We hypothesize that both TR2 and TR4 play significant roles in the regulation of developmental, physiological, and behavioral systems. To determine the specific roles of TR2 and TR4 in vivo, we will employ mouse models exhibiting ablation of either orphan receptor, as well as animals exhibiting simultaneous ablation of both receptors. Specific aim 1: Characterization of TR4 knockout/Beta-gal knockin (TR4 -/-) mice. Specific aim 2: Characterization of TR2 knockoutJBeta-gal knockin (TR2 -/-) mice. We hypothesize this receptor is more important in early stages of development, and that TR2 -/- mice may die pre- or postnatally. Mortality will be investigated by morphological and histological analyses, and animals surviving to adulthood will be assessed for growth rate and fertility. Specific aim 3: Characterization of TR2/TR4 double knockout mice. Analysis of the double knockout animals will follow the approach described in Specific Aim 2, and the phenotypes will be analyzed in comparison to TR2 knockout, TR4 knockout, and wildtype mice. Specific Aim 4: Analysis of the effects of TR4 and/or TR2 ablation on target gene regulation. TR4, TR2, and TR2/TR4 knockout animals will be tools to study the known target genes of these orphan receptors in an in vivo system, and to confirm the physiological significance of the identified regulatory pathways, and provide sources of material for the screening of novel TR2/TR4 target genes. 4a: Determination of effect of TR4 and/or TR2 target gene ablation on known target gene expression. Compare endogenous gene expression, and protein levels of TR4 and TR2 downstream targets in knockout animals versus wildtype controls. 4b: Identifiy novel genes regulated uniquely or differentially by TR4 and/or TR2 through the use of gene microarray technology, we hope to dissect the differences in target gene regulation mediated by TR4 and TR2 and, in this way, further understand the roles of these enigmatic orphan receptors in mammalian development and physiology. In summary, this proposal provides us the first opportunity to study the potential in vivo physiological roles of the TR2 and TR4 orphan nuclear receptors.